Glioblastoma multiforme (GBM) is a deadly human cancer with unacceptably poor therapies. In pilot studies, we found that LC-1, a prodrug of the natural product parthenolide (PTL), penetrates the blood-brain barrier in mice and accumulates to micromolar levels in tissue. Furthermore, oral dosage of LC-1 to mice bearing GL261 murine GBM extended survival by greater than 10-days (p < 0.01) in our system. Unfortunately, the molecular mechanism of LC-1 activity in GBM is not established, which prevents the rational design of more potent and specific analogues. We hypothesize that elucidation of the molecular targets of PTL may reveal an entirely new strategy for attacking GBM. In ongoing studies, we developed a pair of alkyne-tagged PTL analogues that have enabled the determination of PTL target proteins in primary human acute myeloid leukemia (AML) cells. Our probes, `functional' and `non-functional', are iso-structural mimics of PTL with different inhibitory activities to cancr cells. `Functional' PTL-alkyne bears similar growth inhibitory properties as parent natural product PTL, whereas `non-functional' PTL-alkyne does not affect cancer cell growth. When both probes are used in tandem during protein pulldown studies, PTL-bound proteins can be correlated with anti-proliferative activity. In other words, our novel reagents allow us to categorize proteins as either associated with the anti-proliferative activity of PTL (the protein was pulled down by `functional', but not `non-functional' PTL-alkyne) or a non-specific binder (both ``functional' and `non-functional' PTL-alkyne bound the protein). Given that PTL is a covalent natural product, our approach reduces the number of proteins for follow-up validation studies. Our long-term goal is to develop innovative and efficacious therapies to target adult and pediatric GBM. The objective of this application is to elucidate the molecular targets of PTL in GBM. Our central hypothesis is that PTL-alkyne probes will enable the identification of the activity-associated molecular targets of PTL in GBM. To accomplish our goal, we propose the following Specific Aims: (1) Identify the activity-associated protein targets of PTL in GBM and (2) Characterize the activity-associated protein targets of PTL in GBM. In the latter aim, we will especially focus on the characterization of F120A, a reactive oxygen species-modulating enzyme identified as a PTL-target protein from our AML studies, which may confer anti-proliferative activity to GBM cells if similarly engaged. Our research is innovative because we have identified a brain-penetrant small molecule inhibitor of GBM growth with activity in vivo, which is PTL. Furthermore, we will employ our innovative, iso-structural PTL- alkyne analogues to enable identification of the molecular targets of the natural product. Our research is significat because we have the opportunity to elucidate the molecular target of PTL in GBM, which will enable the rational design of more potent analogues. Additionally, our research is significant because we have the opportunity to validate a new strategy for eradicating GBM. Given the lack of effective treatments for adult and pediatric GBM (2-year survival in adults is 26%) any advance in this field will be exceptionally significant.